PROJECT SUMMARY Melanoma is the most lethal subtype of skin cancer with a high frequency of BRAF mutations. Over 90% of BRAF mutations are V600E, which results in hyperactivation of the mitogen-activated protein kinase (MAPK) pathway. Patients with metastatic melanoma have minimal therapeutic options and responses to current strategies targeting BRAF and/or MEK1/2 kinases are only transient due to the emergence of drug resistance. Our lab has identified Cu as a novel vulnerability within the MAPK pathway that can be leveraged to induce antineoplastic activity in BRAFV600E melanomas. Cu binding to MEK1/2 is required for kinase activity, and lowering Cu levels reduced tumorigenesis in a murine model of BRAFV600E metastatic melanoma. While Cu is traditionally thought of as a structural or catalytic cofactor for enzymatic reactions, a newfound role in modulating kinase activity has brought another layer of regulation to signaling pathways that can be further studied and potentially targeted in therapeutic strategies. Interestingly, a Cu chelator, tetrathiomolybdate (TTM), both synergized with inhibitors of BRAF and MEK1/2 and showed effectiveness in inhibitor resistant melanoma cells. However, similar to BRAF and MEK1/2 inhibitors, the response to Cu chelation is cytostatic and short-term and thus, elucidation of additional Cu-dependent kinases may unlock other mechanisms of TTM that can propel this drug to be a more efficacious therapeutic option for late-stage BRAF-driven melanoma. In studying Cu dependent kinases, we identified large tumor suppressor 1/2 (LATS1/2) as targets of Cu chelation. LATS1/2 are integral kinases involved in mediating cell growth within the Hippo pathway. Inhibition of LATS1/2 upregulate oncogenic Yes- associated protein (YAP). Conversely, LATS1/2 are phosphorylated and activated by upstream kinases enabling LATS1/2 to phosphorylate YAP, preventing its nuclear localization. Enhanced YAP nuclear accumulation is observed in cancers including BRAFV600E-mutant melanoma, where it is active to initiate a growth-promoting transcriptional program. Aside from phosphorylation, additional modulators of LATS1/2 kinase activity are unknown. We found that Cu chelation inhibits LATS1/2 kinase activity in vitro and that LATS1/2 bind Cu. However, it is not clear whether Cu is necessary for LATS1/2 kinase activity in vivo. While Cu chelation inhibits oncogenic MAPK signaling, I hypothesize that Cu is needed for LATS1/2 kinase activity and that concomitant inhibition of LATS1/2 will dampen the Hippo pathway, consequently limiting the efficacy of Cu chelation therapy. In Aim 1, I will define the specific Cu binding sites within LATS1/2 to assess the contribution of Cu to LATS1/2 kinase activity in BRAFV600E mutant melanoma cell lines. In Aim 2, I will use genetic and pharmacologic means to co-target YAP and Cu in murine models of melanoma to assess the efficacy of the combination therapy in a BRAFV600E mutant setting and further test this regime in combination with a standard of care BRAF inhibitor vemurafenib. Overall, this study will improve our understanding of Cu regulation of kinases and will provide insight into enhancing Cu chelation as a durable therapeutic option for BRAF-driven melanomas.